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-   -   [NMR paper] The Cu2+-nitrilotriacetic acid complex improves loading of ?-helical double histidine site for precise distance measurements by pulsed ESR (http://www.bionmr.com/forum/journal-club-9/cu2-nitrilotriacetic-acid-complex-improves-loading-helical-double-histidine-site-precise-distance-measurements-pulsed-esr-25411/)

nmrlearner 12-08-2017 01:06 AM

The Cu2+-nitrilotriacetic acid complex improves loading of ?-helical double histidine site for precise distance measurements by pulsed ESR
 
The Cu2+-nitrilotriacetic acid complex improves loading of ?-helical double histidine site for precise distance measurements by pulsed ESR

Publication date: Available online 7 December 2017
Source:Journal of Magnetic Resonance</br>
Author(s): Shreya Ghosh, Matthew J. Lawless, Gordon S. Rule, Sunil Saxena</br>
Site-directed spin labeling using two strategically placed natural histidine residues allows for the rigid attachment of paramagnetic Cu2+. This double histidine (dHis) motif enables extremely precise, narrow distance distributions resolved by Cu2+-based pulsed ESR. Furthermore, the distance measurements are easily relatable to the protein backbone-structure. The Cu2+ ion, has till, now been introduced as a complex with the chelating agent iminodiacetic acid (IDA) to prevent unspecific binding. Recently, this method was found to have two limiting concerns that include poor selectivity towards ?-helices and incomplete Cu2+-IDA complexation. Herein, we introduce an alternative method of dHis-Cu2+ loading using the nitrilotriacetic acid (NTA)-Cu2+ complex. We find that the Cu2+-NTA complex shows a four-fold increase in selectivity toward ?-helical dHis sites. Furthermore, we show that 100% Cu2+-NTA complexation is achievable, enabling precise dHis loading and resulting in no free Cu2+ in solution. We analyze the optimum dHis loading conditions using both continuous wave and pulsed ESR. We implement these findings to show increased sensitivity of the Double Electron-Electron Resonance (DEER) experiment in two different protein systems. The DEER signal is increased within the immunoglobulin binding domain of protein G (called GB1). We measure distances between a dHis site on an ?-helix and dHis site either on a mid-strand or a non-hydrogen bonded edge-strand ?-sheet. Finally, the DEER signal is increased twofold within two ?-helix dHis sites in the enzymatic dimer glutathione S-transferase exemplifying the enhanced ?-helical selectivity of Cu2+-NTA.
Graphical abstract

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